1. Field of the Invention
The present invention relates to a screening method for genes of an industrial yeast used for the production of an alcoholic beverage such as beer or sake, a fuel alcohol, etc. and particularly for genes of brewing yeast used for the production of an alcoholic beverage. More particularly, it relates to a method where, in the production of an alcoholic beverage, DNA sequence information of brewing yeast is compiled in a database so that the gene which participates in increase in productivity and/or improvement in flavor such as stabilization, reinforcement, etc. of the flavor is selected; a method for breeding yeast suitable for the brewing in which expression of a gene is controlled, such as yeast in which the selected gene is disrupted or yeast in which the gene is overexpressed; and a method for the production of an alcoholic beverage using the bred yeast.
2. Description of the Prior Art
Development of techniques for production of fuel alcohols, alcoholic beverages such as beer or sake, etc. has been carried out using industrial yeast. Especially in the production of an alcoholic beverage using brewing yeast, there has seen a brisk development in the techniques for increasing productivity and improving flavor such as stabilization or enhancement of flavor of an alcoholic beverage.
The most consumed alcoholic beverage in the world is beer and the amount of beer produced in the world in 2001 was about 140,000,000 kL. Type of beer is roughly classified into three depending upon type of yeast and fermentation method. The three types are, naturally fermented beer where fermentation is carried out utilizing yeast and microorganisms inhabiting in breweries; ale-type beer where fermentation is carried out using a top fermenting yeast belonging to Saccharomyces cerevisiae (hereinafter, abbreviated as S. cerevisiae) at the temperature of 20 to 25° C. and the following aging period is shortened; and lager-type beer where fermentation is carried out using a bottom fermenting yeast belonging to Saccharomyces pastorianus at the temperature of 6 to 15° C. and then subjected to a low-temperature aging. At present, not less than 90% of the beer produced in the world is a lager-type beer and, therefore, the bottom fermenting yeast that is used for brewing of the lager-type beer has been most widely used in beer brewing.
In the so-called fermentation production where production is carried out using a microorganism including the above-mentioned brewing yeast, it is important that the fermentation process is optimized and that the useful strain is selected and bred, in order to increase productivity and improve quality of the product.
In the case of optimization of beer brewing, there has been conducted a method where an amount of yeast metabolites such as alcohol (e.g. ethanol), ester, organic acid, etc. are monitored, and then temperature, quantity of airflow, content of raw material, etc. are controlled. In such a case, material uptake and excretion by yeast cells and metabolism in the cells are handled as a black box and only very superficial control is carried out. In addition, for the purpose of giving, for example, high flavor to an alcoholic beverage, a process control method for suppressing the amount of oxygen supply during beer brewing or the like has been tried. In such a method, however, growth rate of the yeast itself is reduced due to insufficient oxygen, and adverse effect such as retardation of fermentation and/or deterioration of beer quality may arise. Accordingly, there has been a limit on the improvement in productivity and quality of beer by means of optimization of fermentation processes.
On the other hand, with regard to a method of breeding useful industrial yeast such as useful beer yeast, a technique for selecting desirable strain has been widely used rather than actual breeding. Beer brewing per se has been performed since well before the discovery of microorganisms by Pasteur and, in the beer brewing, a method of selecting more suitable strain of beer yeast from many strains of yeast used in the beer brewery has been traditionally carried out while there have been few cases where beer yeast with good traits is positively bred.
As an example of a positive breeding method, there is a method where artificial mutagenesis by chemicals or radioactive rays is used. However, brewing yeast, particularly a bottom fermenting yeast which is widely used in beer brewing, is in many cases a polyploid. In that case, it is not possible to produce the desired mutant unless mutation takes place in all of the alleles to be mutated. Accordingly, although it is possible to induce desirable mutation in the case of a haploid laboratory yeast, it is substantially impossible in the case of beer yeast which is a polyploid.
In recent years, there has been tried a breeding where mutation or cross-breeding is carried out by using spores isolated from bottom fermenting yeast (c.f., for example, Non-Patent Document 1). However, the bottom fermenting yeast is a polyploid, and has complicated chromosome structure, therefore, isolation of spores having proliferation ability is difficult, and moreover it is almost impossible to obtain a strain with good traits therefrom.
On the other hand, it has recently become possible that desired genes are introduced and expressed in the brewing yeast using a genetic engineering technique, whereby it has become possible to breed yeast with the desired character by using the results of functional analysis of genes and the genes which have been functionally analyzed. However, as compared with the baker's yeast (S. cerevisiae; c.f., for example, Non-Patent Document 2) of which the whole genome sequence is already clarified, the whole genome sequence of the bottom fermenting yeast has not been clarified and there have been only a very few findings about the gene participating in brewing character specific to bottom fermenting yeast and about the function of the said gene in beer brewing.
In recent years, transcriptome analysis has been conducted using DNA microarray where DNA fragments or nucleotide oligomers, each of which has a partial sequence of structural gene or internal region of the chromosome are fixed on solid support. For example, Olesen, et al. conducted a comprehensive genetic expression analysis of bottom fermenting yeast during the brewing using GeneFilters (manufactured by Research Genetics Co.) (c.f., for example, Non-Patent Document 3). However, since the whole genome sequence of bottom fermenting yeast has not been clarified yet, it is ambiguous that what gene is monitored for its expression precisely. As a result, such information is quite insufficient to apply to metabolic analysis of bottom fermenting yeast, and to breeding of useful yeast, and to control of beer brewing process.
At present, the whole genome sequences of more than 100 species of microorganisms have been determined (c.f., for example, Non-Patent Document 6) including S. cerevisiae, Escherichia coli (c.f., for example, Non-Patent Document 4) and Mycobacterium tuberculosis (c.f., for example, Non-Patent Document 5). On the basis of the determined DNA sequences, genes of these microorganisms are identified and function of an enormous number of genes have been predicted without conducting genetic, biochemical and molecular biological experiments. However, industrial yeast such as brewing yeast which has high ploidy and complicated chromosome structure, and thus an assembly (an operation for connecting the DNA sequences) is presumed to be difficult. Therefore, the whole genome sequence of bottom fermenting yeast which contains two different types of genome (c.f., for example, Non-Patent Document 7) has not been reported yet.
In the production of specific alcohols or alcoholic beverages, there is a technique to increase concentration of sulfite in the product for control of flavor. Sulfite is known as a compound which has anti-oxidative activity, and has been widely used as an antioxidant in the fields of food, beverage and pharmaceuticals, and also in an alcoholic beverage. For example, in the case of wine that requires a long aging period, sulfite plays an important role for the preservation of its quality. It is also known that, in beer brewing, the quality preservation period becomes long in accordance with the increase in concentration of sulfite contained in the product. Thus, when the amount of sulfite in the product is increased, it is possible to prepare a product that has excellent flavor stability and a long quality preservation period.
The simplest way to increase the amount of sulfite in the product is addition of sulfite. In Japan, so far as wine is concerned, it is permitted by the Ministry of Health, Labor and Welfare to add sulfite to an extent of not more than 350 ppm in terms of residual sulfite concentration. In that case, however, since sulfite is categorized as food additives, it is not appropriate to add sulfite to beer when a negative image of consumers to food additives is taken into consideration.
However, the yeast used in brewing produces hydrogen sulfide by the reduction of sulfate in the medium in order to synthesize sulfur-containing metabolites such as sulfur-containing amino acids. Sulfite is an intermediate metabolite of this pathway. If sulfite is efficiently excreted outside the cells during fermentation period, the amount of sulfite both in the wort and in the product can be increased.
There are two methods for increasing sulfite concentration in the wort during fermentation. One is control of fermentation process and another is breeding of brewing yeast. As for control of fermentation process, amount of sulfite produced during fermentation is inversely proportional to the concentration of dissolved oxygen and, therefore, there has attempted, a method where the concentration of dissolved oxygen is reduced so that amount of sulfite is increased and at the same time the oxidation of sulfite is suppressed. However, in that method, growth rate of yeast is reduced due to lack of oxygen, which has negative effects such as retardation of fermentation and deterioration of quality. Therefore that method is not practical.
On the other hand, as mentioned above, a genetic engineering technique has been developed for breeding brewing yeast. For example, there are some reports focused on sulfur metabolism of yeast. Sulfite (SO2) is an intermediate product of sulfur-containing amino acid and vitamin synthesis and is produced via a pathway of sulfate ion (SO42−)→APS (adenyl sulfate)→PAPS (phosphoadenylyl sulfate)→sulfite ion (SO32−) where the sulfate ion is incorporated from outside of the cells. There is an attempt that copy numbers of MET 3 gene participating in the reaction of sulfate ion (SO42−)→APS (adenylyl sulfate) and of MET 14 gene participating in the reaction of APS (adenylyl sulfate)→PAPS (phosphoadenylyl sulfate) are increased to improve the ability of the yeast for the production of sulfite (c.f., for example, Non-Patent Document 8). There is another example of an attempt where reduction of sulfite ion (SO32−) is inhibited by disruption of MET 10 gene whereby amount of sulfite produced by the yeast is increased (c.f., for example, Non-Patent Document 9). According to such attempts, amount of sulfite produced by an MET 10 gene disruptant is increased to an extent of not less than ten-fold of the parental strain, but on the other hand, some retardation in fermentation and increase in the amounts of acetaldehyde and 1-propanol in the beer are noted, which has become a problem for the practical use.
Accordingly, although development of breeding methods for industrial yeast such as brewing yeast using genetic engineering have been in progress, it is the current status that, due to insufficient genomic information of brewing yeast, selection of the gene participating in a brewing character of brewing yeast, analysis of function of protein encoded by the gene and utilization of those findings for breeding have not been sufficiently carried out.
Thus, a method for breeding yeast which shows the desired character without deterioration of fermentation speed and product quality has not been established yet and there has been a big demand for the development of such a method not only in the brewing industry but also in the industries where yeast is used.
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